PROJECT SUMMARY Rotaviruses are an important cause of diarrheal disease. While rotavirus species A (RVA) causes endemic diarrheal disease in infants and children, rotavirus species B (RVB) causes sporadic epidemic diarrheal disease primarily affecting adults. Between RVA and RVB, only the NSP1 genome segment is predicted to encode non-homologous proteins. For RVB, this segment encodes two proteins of unknown function. A lack of knowledge of NSP1-encoded protein function, coupled with the lack of an RVB tissue culture model, has impeded studies of RVB biology. We recently discovered that the smaller protein encoded by the RVB NSP1 segment, NSP1-1, is a fusion-associated small transmembrane (FAST) protein whose expression results in syncytia formation in a host species-specific manner. We also found that RVB NSP1-1 expression promotes RVA replication. FAST proteins are modular viral nonstructural proteins that are expressed by other Reoviridae viruses and contribute to viral replication and pathogenesis. To overcome the lack of an RVB culture model, we will use RVA reverse genetics technology to elucidate biological functions of NSP1-1 in the context of viable chimeric rotaviruses. Building on our preliminary findings, we propose two integrated specific aims to test the hypothesis that rotavirus NSP1-1 is a modular tropism determinant that promotes direct cell-cell spread. FAST proteins contain N-terminal, transmembrane, and C-terminal domains. Our preliminary studies suggest RVB NSP1-1 shares this domain organization and demonstrate that human RVB NSP1-1 mediates fusion of primate cells but not rodent cells. In Specific Aim 1, we will test the hypothesis that NSP1-1 is a modular FAST protein that functions in a host species-specific manner. We will determine the capacity of NSP1-1 from different rotavirus species or strains to mediate fusion of cultured cells derived from distinct host species. We will identify the domain responsible for species-specificity by exchanging domains between human RVB NSP1- 1 and p10, a FAST protein that mediates rodent cell fusion. These studies will identify regions of NSP1-1 that may limit rotavirus zoonotic transmission. In Specific Aim 2, we will test the hypothesis that NSP1-1 enhances viral replication and spread. We will engineer RVB NSP1-1-expressing RVA reporter viruses using recently- developed reverse genetics technology and quantify their replication and spread in the presence of fetal bovine serum. Fetal bovine serum inhibits rotavirus entry via standard pathways, thereby permitting virus spread primarily via direct cell-cell fusion. These studies will reveal functions of NSP1-1 during viral infection and generate hypotheses regarding its contributions to pathogenesis. Together, these discoveries will provide insight into epidemiological differences between RVA and RVB. Furthermore, engineering chimeric rotaviruses that package segments from divergent species will promote future studies of other rotaviruses for which we lack culture models and uncover concepts broadly applicable to segmented, dsRNA virus engineering.